Picornaviruses are small non-enveloped positive strand RNA viruses that can cause a wide range of clinical manifestations in humans and animals. Based on a number of properties including sequence homologies and acid sensitivity, Picornaviruses are separated into a number of genera among them are many important pathogens of humans and animals.
Picornaviruses have naked nucleocapsid. The capsid is an arrangement of 60 protomers in a tightly packed icosahedral structure. Each protomer consists of 4 polypeptides known as VP (viral protein) 1, 2, 3 and 4. VP2 and VP4 polypeptides originate from one precursor known as VP0, which is cleaved after the internalization of the viral genomic RNA into the cell. VP4 is located on the internal side of the capsid. Depending on the type and degree of dehydration the viral particle is around 27-30 nm in diameter.
Picornaviruses have a monopartite, linear, polyadenylated ssRNA(+) genome of 7.1-8.9 kb, that is composed of a single ORF encoding a polyprotein. Viral genomic RNA has a viral protein (VPg) at its 5′ end instead of a methylated nucleotide cap structure. The long UTR at the 5′ end contains an internal ribosome entry site (IRES). The P1 region encodes the structural polypeptides. The P2 and P3 regions encode the nonstructural proteins associated with replication. The shorter 3′ UTR is important in (−)strand synthesis. L is an additional N-terminal leader protein present in some genera that can either be a protease (aphthoviruses, erboviruses) or have other function (kobuvirus, cardiovirus).
The virion RNA is infectious and serves as both the genome and viral messenger RNA. The IRES allows direct translation of the polyprotein. The polyprotein is initially processed by the viral protease(s) into various precursor and mature proteins to yield the structural proteins, replicase, VPg, and a number of proteins that modify the host cell, ultimately leading to cell lysis.
Enterovirus 71 (EV71) is a member of the Picornaviridae family of single stranded RNA viruses. It is a non-enveloped virus and its capsid is constituted of multiple coat proteins produced as fragments of a single viral translation product. The processing of viral polyprotein into structural and non-structural components is presented in FIG. 1 (prior art). The P1 region of the polyprotein gene encodes the structural proteins while P2 and P3 regions encode non-structural components of the virus. After cleavage of the structural protein precursor P1 (1ABCD in FIG. 1) from the polyprotein by the viral protease 2A, the P1 precursor is processed into the capsid proteins VP0, VP1 (1D fragment in FIG. 1) and VP3 (1C fragment in FIG. 1). The 3C component and its precursor 3CD—encoded by the P3 region—are the viral proteases responsible for processing the P1 precursor into capsid proteins. The VP0, VP1 and VP3 protomers spontaneously assemble into empty capsids and it is believed that viral RNA is packaged into the particles after the assembly of empty particles. Association of the empty capsid with genomic RNA results in a structural shift, internalization of the RNA, autocatalytic cleavage of VP0 into VP2 (1B fragment in FIG. 1) and VP4 (1A fragment in FIG. 1), and maturation into a stable 150S virion. Empty capsids, containing the uncleaved VP0 precursor, are commonly found during picornavirus infections.
Production of EV71 VLPs in insect cells has been obtained from the co-expression of the P1 precursor protein with the 3CD protease (Hu et al., 2003, Biotechnology Letters 25: 919-925). Use of a single baculovirus vector for the production of P1 and 3CD is described by Chung et al. (2008, Vaccine 26: 1855-1862) Immunogenicity studies in mice showed that purified EV71 VLPs conferred protection to a challenge with lethal doses of the virus.
The VP1 protein from EV71 has been produced in fruits of transgenic tomatoes, and feeding mice with transgenic fruit containing VP1 resulted in the development of VP1-specific fecal IgA and serum IgG (Chen et al., 2006, Vaccine 24: 2944-2951).
The P1 precursor protein and protease 3C of the foot and mouth disease virus (FMDV) was co-expressed in transgenic alfalfa (Dus Santos et al. 2005, Vaccine 23: 1838-1843). The alfalfa was stably transformed with a single vector comprising the genomic region of FMDV P1 (1A, 1B, 1C, 1D), 2A, the first 16 amino acid residues of the N terminus of 2B, the complete sequence of 3B1, 3B2, 3B3, 3C and the first 16 amino acid residues of the N terminus of 3D. Immunogenicity of crude protein extracts from the transgenic plants was demonstrated by intraperitoneal administration in Balb/c mice. Immunized mice were also protected against a lethal FMDV challenge. The levels of antigen expression were low for practical purposes.
Argentinean Application AR078257 discloses a transgenic plant expressing an empty capsid virus, wherein the transgenic plant comprises in its genome a DNA construct encoding a P1 precursor polypeptide linked to autocatalytic 2A protease. The DNA construct may further contain protein fragment 2B attached to the sequence encoding the 3C protease linked to a fragment of the sequence encoding a protein fragment 3D.